Multiphor 2-D electrophoresis

Litta Olsen litta.olsen at sars.uib.no
Mon Mar 30 02:24:51 EST 1998


Hello Matthias.

Thank you for your reply. It is nice to talk to someone working with the
same stuff.

First:
There should not be a problem with the quantity of my proteins, nor with
a particular one.
I use the buffer described in the manual for the reswelling tray and it
is almost the same as the one you mentioned except that the DTT is 20
mM.
I did have a problem with the current limiting the voltage during the
first two phases of IEF. This is probably due to ions in my sample, and
I can imagine that this is a problem for others, too, since one is
supposed to be able to reswell 100 µl of sample into one strip.  
Perhaps you do not use this way to apply the samples? I know that the
system with this reswelling tray is a new approach. 
Do you think it would be better to load the samples in sample cups ? It
is definately better when you think of protease activity.

Secondly:
The company suggested that I raise the temperature during 2.dimension.
It could be between 15 and 20 degrees. Too low temperature could cause
the urea to fall out and the proteins enter the gel differently, but
this should only cause a general uneven frontline and not a smiling
effect. To reduce smiling I simply tried to put the two strips as
centered as possible, so that the distance from the edge of the gel to
the beginning of the strips is as big as possible. The problem is caused
by lower current at the ends of the buffer strips, which I, by the way,
find being a bit short. You can cut off some of the plastic support on
the strips and minimize the distance between them in order to increase
the unused space on each side of gel.

I noticed that the mail adress is not in your name. Can I mail you on
this anyway ?

Litta



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